In recent years, various techniques which are highly sensitive and enable assaying trace amounts of samples have been proposed for investigating the structure-activity relationships of complex or conjugated carbohydrates (glycoconjugate) such as glycoproteins.
For example, a method of microanalysis of sugar chains comprises tritiating on the occasion of conversion of the reducing terminus to a sugar alcohol group and utilizing the radioactivity of tritium. The detection sensitivity is good, namely at the picomole level. However, since tritium is a radioactive substance, various restrictions are inevitable.
A high-sensitivity assay method free of such restrictions is known, which uses an organic compound such as 2-aminopyridine as a fluorescent labeling agent [e.g. S. Hase et al., Journal of Biochemistry, 95, 197-203 (1984)].
This method comprises reacting the reducing terminus of a carbohydrate or a sugar chain with the 2-amino group of said organic compound, reducing the resulting Schiff base and detecting the product by its fluorescence. It is disclosed that the sensitivity is at the picomole level, like in the above case.
The above fluorescent labeling agent is effective in the structure analysis of carbohydrate chains but, because of its unifunctionality, cannot cause formation of conjugates of carbohydrates with other biological substances such as proteins, amino acids, lipids and nucleic acids. Therefore, it is not fully satisfactory for global analysis.